Using Truncated Guide RNAs (tru-gRNAs) to Increase Specificity for RNA-Guided Genome Editing

Patent metadata

Field	Value
Publication number	US-2020071730-A1
Application number	US-201916572248-A
Country	US
Kind code	A1
Filing date	Sep 16, 2019
Priority date	Mar 15, 2013
Publication date	Mar 5, 2020
Grant date	—

Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

First claim

1 .- 28 . (canceled)

29 . A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell:
a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence present on a double-stranded DNA molecule, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is:
(i) a single guide RNA that includes a complementarity region at the 5′ end of the single guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or
(ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA, and
wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the target region of the double-stranded DNA molecule, thereby modifying the target region of a double-stranded DNA molecule in a cell, and wherein the dCas9-HFD comprises a heterologous functional domain (HFD) that modifies gene expression, histones, or DNA.

30 . The method of claim 29 , wherein the HFD is a transcriptional activation domain, an enzyme that catalyzes DNA demethylation, an enzyme that catalyzes histone modification, or a transcription silencing domain.

31 . The method of claim 30 , wherein the transcriptional activation domain is from activator domain VP64 or NF-kappa B subunit p65 (NF-κB p65).

32 . The method of claim 30 , wherein the enzyme that catalyzes histone modification is lysine-specific histone demethylase 1 (LSD1), a histone methyltransferase (HNMT), histone acetyltransferase (HAT), histone deacetylase (HDAC), or histone demethylase.

33 . The method of claim 30 , wherein the transcription silencing domain is from Heterochromatin Protein 1 alpha (HP1α) or Heterochromatin Protein 1 beta (HP1β).

34 . The method of claim 29 , wherein the target region is in a target genomic sequence.

35 . The method of claim 29 , wherein the cell is a eukaryotic cell.

36 . The method of claim 35 , wherein the cell is a mammalian cell.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Sander Jeffry D
• Maeder Morgan
• Fu Yanfang

Classifications

• C12N2710/00033

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• C07K14/195

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• C12Y114/11

  with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11) · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12Y301/21004

  Type II site-specific deoxyribonuclease (3.1.21.4) · CPC title